Available online at www.sciencedirect.

com

MEAT SCIENCE
Meat Science 80 (2008) 370379 www.elsevier.com/locate/meatsci

The eects of extended curing on the microbiological, physicochemical and sensorial characteristics of Cecina de Leon
Cristina Molinero a, Beatriz Martnez a,*, Begona Rubio a, Jordi Rovira b, Isabel Jaime b
a

Estacion Tecnologica de la Carne, Instituto Tecnologico Agrario de Castilla y Leon, C/ Filiberto Villalobos s/n, Apartado de Correos no 58, Salamanca, 37770 Guijuelo, Spain b Departamento de Biotecnologa y Ciencia de los Alimentos, Universidad de Burgos, Plaza Misael Banuelos s/n, 09001 Burgos, Spain Received 12 July 2007; received in revised form 25 October 2007; accepted 20 December 2007

Abstract The objective of this study was to evaluate the eects of curing times on the characteristics of 7-month dry-cured beef cecina stored for up to 12-months at 16 C and 65% relative humidity. Microbiological and physicochemical parameters, sensorial properties and consumer preferences were analysed at three dierent processing times (210, 270 and 360 days). Curing time signicantly aected (p < 0.05) most of the parameters studied. Moisture and aw decreased (p < 0.05) and NaCl content increased from day 210 to day 360, whereas microbial counts decreased (p < 0.05) from day 210 to day 360. The continued increase of amino acid content and free fatty acid (p < 0.05) until day 360 contributed to modications in the characteristics of the nal product. Thus, cecina with longer processing times had higher scores for colour, avour and aftertaste. Consumer preferences indicated that the sensory quality of cecina improved from day 210 to day 270 of processing, after which no further changes were noted as curing was extended to 360 days. 2008 Elsevier Ltd. All rights reserved.
Keywords: Dry-cured cecina; Curing; Physicochemical parameters; Sensory properties; Consumer preferences

1. Introduction Spanish Cecina de Leon is a salted, dried and smoked beef meat product, traditionally manufactured in the region of Leon (North-western Spain) where it is of great economic importance. This product has a typical red colour, smoked avour and a slightly salty taste. Its peculiar characteristics have made Cecina de Leon a highly palatable product that is widely accepted by consumers. It carries the Protected Geographical Indication (PGI) label, which dierentiates this original product from other types of cecina. Nowadays, a wide variety of dry-cured meat products are produced depending on the raw materials and process ing conditions (Toldra, 2006). In general, important reductions in both moisture content and water activity take place
*

Corresponding author. Tel.: +34 923580688; fax: +34 923580353. E-mail address: mardomma@itacyl.es (B. Martnez).

throughout the elaboration process of dry-cured meat products. This reduction can vary depending on drying conditions, and decreased water activity may aect enzyme activity, which inuences the sensorial characteristics of the nal product (Toldra, 2006). Throughout the processing of dry-cured products, proteolysis and lipolysis constitute two of the most important mechanisms with consequences for nal sensory quality. Proteolytic events have been shown to be an important source of aroma and avour, as they release several compounds related to avour development, such as free amino acids (Cordoba et al., 1994). Lipolysis also plays an important role in the development of sensorial characteristics, because it causes an increase in free fatty acid content, and also catalyses other reactions such as oxidation leading to the release of a large number of volatile compounds responsible for the characteristic avours of certain foods (Yang, Ma, Qiao, Song, & Du, 2005).

0309-1740/$ - see front matter 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2007.12.023

C. Molinero et al. / Meat Science 80 (2008) 370379

371

When processing is over longer periods of time, the action of muscle proteases and lipases is more intense (Toldra, 2006), for which reason traditional dry-cured products have always been prepared with a nal maturation/ripening step in a cellar. Studies on ham ripening suggest that proteolysis and lipolysis contribute to improving ham quality (Cilla, Martnez, Beltran, & Roncales, 2005). However, evidence exists to suggest that if modications are too severe the quality could decrease due to the development of a defective texture related to over-ripening because of uncontrollable proteolytic processes (Arnau, Guerrero, & Gou, 1997; Cilla et al., 2005; Parolari, 1994; Virgili, Parolari, Schivazzappa, Soresi Bordini, & Borri, 1995). In modern processing, the cellar stage is carried out in drying rooms under controlled conditions of temperature and relative humidity. The minimal dry-curing time indicated by the PGI Cecina de Leon is seven months following salting. The aim of this study was to evaluate the physicochemical, microbial and sensory characteristics of cecina at three dierent processing times (210, 270 and 360 days) in order to investigate the inuence of extended curing on its properties. 2. Materials and methods 2.1. Manufacture of cecina The manufacturing process closely followed the speci cations of the PGI Cecina de Leon (BOCyL, 1994). Twelve knuckles (Quadriceps femoris composed of the muscles Rectus femoris and Vastus lateralis, V. medialis and V. intermedius), each with an average weight of 12.05 0.05 kg, were selected and the pieces were trimmed to a uniform shape by removing external connective tissue. The pieces were then covered in salt and placed in piles formed of layers of meat and salt. They were held at 34 C and a relative humidity (RH) of 8590% in a chamber for 3 days. Next, the pieces were washed to remove excess salt and held at 34 C and 8590% RH for another 50 days (post-salting or salting equalization stage). After the post-salting stage, the pieces were smoked using oak wood for 5 days at 1215 C and 6575% RH in a smoke house. They were subsequently dried for 5 months at 1015 C and at an RH that was progressively reduced from 80% to 65% in a room with controlled temperature and RH. Then, four pieces were sampled and the rest were held at 1516 C and 6560% RH for 2 months (4 pieces) and 5 months (4 pieces). The environmental conditions (temperature and RH) were monitored throughout processing. The air convection in the drying room was intermittent and the air velocity around the pieces when the fan was running ranged between 0.3 and 0.6 m/s. Samples were taken from two whole pieces at day 210 (corresponding to 7 months), at day 270 (corresponding to 9 months) and at day 360 (corresponding to 12 months) for analysis of microbial counts, pH and aw values, moisture content, instrumental

measurement of colour and texture and descriptive sensory analysis. The rest of the pieces were minced, vacuum packed and stored at 80 C until protein, fat, NaCl, carbohydrate and hydroxyproline content, as well as proteolysis and lipolysis-related parameters were analysed. Then, two whole pieces at day 210 and two at day 270 were packed and stored at 4 C until the end of the process (360 days) for triangle and preference tests. 2.2. Microbiological analyses Ten grams of cecina were homogenized with 90 ml of tryptone water (Scharlau, Spain) for 2 min in a sterile plastic bag in a PK 400 Masticator (IUL, S.A., Barcelona, Spain). Serial decimal dilutions were made in sterile tryptone water and in duplicate, 1 ml or 0.1 ml samples of appropriate dilutions were poured or spread onto total count and selective agar plates. The following microbiological analyses were performed: total viable counts were determined on Plate Count Agar (Scharlau, Spain) incubated at 30 C for 48 h; Micrococcaceae on Mannitol Salt Agar (Sharlau, Spain) incubated at 37 C for 48 h; lactic acid bacteria on Man Rogosa Sharpe Agar (Sharlau, Spain) incubated at 30 C for 48 h; and enterobacteria on Violet Red Blue Glucose Agar (VRBGA, Oxoid, Spain) incubated at 37 C for 24 h. 2.3. Physicochemical analyses The pH was determined with a Crison 2001 pH meter (Crison Instruments S.A., Barcelona, Spain) equipped with a puncture electrode. Water activity (aw) was measured using a Decagon CX-2 AQUALAB hygrometer (Decagon Devices Inc., Pullman, WA, USA). Moisture, fat and protein were determined according to the ISO methods 1442:1997 (ISO, 1997), 1443:1973 (ISO, 1973) and 937:1978 (ISO, 1978) with a nitrogen to protein conversion factor of 6.25. NaCl was determined following ISO methods 1841-1:1996 (ISO, 1996). Total carbohydrates were quantied by the Lu-Schoorl method (B.O.E., 1979). Determination of hydroxyproline content was as described by Bonnet and Kopp (1984). 2.4. Proteolysis-related parameters Non-protein nitrogen (NPN) was determined using the Kjeldahl method 937:1978 (ISO, 1978) on an extract obtained following the procedure proposed by De Ketelaere, Demeyer, Vandekerckhove, and Vervaeke (1974). Amino acid nitrogen (AN) was determined from the 0.6 N perchloric acid soluble fraction of cecina after peptide precipitation with sulfosalicilic acid l0%, according to Moore and Stein (1954). Peptide nitrogen (PN) was quantied by the dierence between the amino acid nitrogen content, after hydrolysis of peptides with 6 N chloride acid of the 0.6 N perchloric acid soluble fraction of cecina and the previously determined AN.

372

C. Molinero et al. / Meat Science 80 (2008) 370379

2.5. Lipolysis-related parameters Total intramuscular lipids were extracted as described by Bligh and Dyer (1959). Neutral lipid, free fatty acids and phospholipids (NL, FFA and PL) from intramuscular fat were separated using NH2-aminopropyl mini-columns as described by Garca-Regueiro, Gilbert, and Daz (1994). Boron triuoride/methanol was used for the preparation of fatty acid methyl esters (Morrison & Smith, 1964). The fatty acid composition of each fraction was determined by gas liquid chromatography of the previously prepared methyl esters. The fatty acids of each fraction were quantied using tridecanoic, nonadecanoic and phosphopentanoic acid as internal standards of neutral lipids, free fatty acids and phospholipids. Gas chromatography using an Omegawax 320 capillary column was carried out using an Agilent 68890 N system, equipped with an on-column injector and a ame-ionization detector. Column temperature was maintained at 200 C throughout the entire run (60 min). The temperatures of the injector and the detector were respectively 250 C and 260 C. Identication of fatty acids was performed by comparison of the retention times with those of known fatty acids and the results expressed as a percentage of the fatty acids of each fraction with respect to total fatty acid content. 2.6. SDSPAGE electrophoresis Myobrillar protein preparation was performed as described by Muroya, Nakajima, Oe, and Chikuni (2006) with some modications. Myobrillar proteins were extracted by homogenizing 200 mg of cecina with 10 ml isolating buer: 20 mM potassiumphosphate buer (pH 6.8) containing 100 mM KCl, 1 mM MgCl2, 1 mM EDTA, 1 mM 2b-mercaptoethanol. Following centrifugation at 2060 g for 15 min, the pellets were solubilized in 10 mM TrisHCl (pH 8.0) containing 5 mM EDTA, 1% SDS, and 40 mM dithiothreitol. The samples were mixed with the sample buer to give a nal concentration of 4 mg/ ml. The sample buer for SDSPAGE contained: 1.6 ml of 10% SDS, 1 ml of 0.5 M TrisHCl, 0.2 ml bromophenol blue, 0.4 ml of 2b-mercaptoethanol and 0.8 ml of glycerol, pH 6.8. The samples were boiled in a water bath for 5 min. The myobrillar proteins obtained were identied by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE) using acrylamide concentrations of 6.5% for stacking gel and 20% for resolving gel (Laemmli, 1970). The slot of the electrophoresis gel was loaded with 15 ll of the myobrillar protein solution. In addition, protein standards (Bio-Rad) were simultaneously run for protein identication. Myosin (200 kDa), b-galactosidase (116.25 kDa), phosphorelase (97.40 kDa), bovine serum albumin (66.20 kDa), ovoalbumin (45 KDa), carbonic anhydrase (31 kDa) and soybean trypsin inhibitor (21.50 kDa) were used as standards. Electrophoresis was performed in a Protean IIxi cell (Bio-Rad) and run at 100 V at 4 C, until the dye track reached the end of the

gels. The gels were stained for 2 h with Coomassie Brilliant Blue R-250, (0.10 g/l) in aqueous ethanol (25%) and acetic acid (10%) and destained in a solution of aqueous ethanol (25%) and acetic acid (8%). After destaining, the proteins were identied according to their molecular weights estimated from their relative mobilities compared to the molecular weight standards (Bio-Rad SDSPAGE molecular). The amount of each protein band was related to the optical density using a densitometry scanner and Quantity One software (Bio-Rad). The most signicant bands of each lane were selected to calculate the relative quantity with respect to the total optical density of each lane. 2.7. Instrumental measurement of colour The surface colour of cecina was measured using a reectance spectrophotometer (Minolta CM-2002; Osaka, Japan) at the three set processing times: 210, 270 and 360 days. The illuminant was D65 (colour temperature of 6504 K) and the standard observer position was 10. Colour results were determined in the CIE-LAB system (CIE, 1976) and lightness (L*), redness (a*, red M green) and yellowness (b*, yellow M blue) were calculated. The pieces of Cecina de Leon were cut in half and colour measurements were performed at room temperature on the cut surface at four dierent locations. 2.8. Instrumental measurement of texture Instrumental Texture Prole Analysis (TPA) (Breene, 1975) was performed with a texture analyzer TA-XT2 (Stable Micro System, Haslemere, UK) at 210, 270 and 360 days. The Stable Micro Systems Texture Expert computer program (version 1.20, Spanish) was used for data collection and calculations. Six cylinders of cecina (2.5 1 cm), extracted from the centre of each piece, were compressed twice with a cylindrical probe of 2.5 cm diameter, at a rate of 1 mm/s. The compression level was 60% of the sample thickness. All tests were performed at room temperature and the parameters determined from the forcetime curves were hardness, springiness, cohesiveness and chewiness. Hardness was dened by the peak force during the rst compression cycle and expressed in g. Springiness was dened as a ratio of time recorded between the start of the second area and the second probe reversal to the time recorded between the start of the rst area and the rst probe reversal. Cohesiveness was calculated as the ratio of the area under the second curve to the area under the rst curve. Finally, chewiness, expressed in g, was evaluated by multiplying hardness, springiness and cohesiveness. 2.9. Sensory evaluation The cecina samples were evaluated by an experienced 8member sensory panel. Eight training sessions were held to familiarise the panellists with the attributes to be evaluated

C. Molinero et al. / Meat Science 80 (2008) 370379

373

and the assessment scale. A quantitative descriptive analysis (UNE 87024-1, 1995; UNE 87024-2, 1996) was used to describe cecina at 210, 270 and 360 days of processing. The attributes were divided into visual aspects (colour homogeneity and colour intensity, marbling, yellowness and intermuscular fat), odour intensity, textural parameters (hardness, chewiness, juiciness and pastiness) and avour characteristics (avour intensity and aftertaste). Descriptors were scored on a scale of 15, where 1 was the minimum and 5 the maximum intensity. A portion of each piece of cecina was presented to the panellists for evaluation of visual parameters and odour intensity, and slices (1.5 mm thick) were presented at room temperature (21 2 C) for evaluation of the remaining sensorial parameters. Unsalted bread and water were served between successive samples. Then, in order to know whether consumers could distinguish between cecina cured for dierent periods of time, 100 habitual consumers of cecina carried out a triangle (UNE 87006, 1992) and preference (UNE 87023, 1995) test. The consumer panel consisted of a variety of students, housewives and sta of dierent organizations. The tests were carried out at three locations in Castile and Leon at various morning sessions. Three triangle tests were conducted: (1) between cecina processed for 210 and 270 days, (2) between cecina processed for 270 and 360 days and (3) between cecina processed for 210 and 360 days. Samples consisting of cecina slices of 1.5 mm of thickness were obtained with a slicing machine and served on plastic plates to participants. The slices were kept at environmental conditions (21 2 C) for at least half an hour before tasting. Finally, a preference analysis between cecina processed for 210, 270 and 360 days was performed by consumers.
Table 1 Microbial counts (log cfu/g) (mean standard deviation) of dry-cured cecina at three dierent processing times (210, 270 and 360 days) Days 210 270 360
a,b,c

The preference ranking test awarded 1 to the least preferred sample and 3 to the most preferred sample. Samples were prepared in the same way as in the triangle tests. 2.10. Statistical analysis A one-way analysis of variance (ANOVA) was used to determine if the ripening time had a signicant inuence on the characteristics of cecina. The means were separated by Tukey-honest signicant dierence at a 5% level. Principal component analysis was performed on the physicochemical, microbiology and sensory data. Parameter correlations were calculated with the Pearson correlation matrix and data analyses were conducted using the Statgraphics Plus 4.0 computer package. 3. Results 3.1. Microbiological parameters Table 1 shows the means and standard deviations of the total viable counts, Micrococcaceae, lactic acid bacteria and enterobacteria counts at 210, 270 and 360 days of processing. Between day 210 and day 360, total viable count, Micrococcaceae and lactic acid bacteria decreased (p < 0.05), possibly due to the inhibitory eect of the low aw values (Vilar, Garca-Fontan, Prieto, Tornadijo, & Carballo, 2000). In fact, positive and signicant correlations were found between aw and total viable count (r = 0.60, p < 0.05), Micrococcaceae (r = 0.94, p < 0.05) and lactic acid bacteria (r = 0.88, p < 0.05); and also between moisture and total viable count (r = 0.78, p < 0.05), Micrococcaceae (r = 0.90, p < 0.05), lactic acid bacteria (r = 0.83, p < 0.05). In general, the counts obtained in this study were lower than those found by Rubio et al. (2007) in Cecina de Leon cured for 11 months. No enterobacteria were detected. 3.2. Physicochemical parameters The means and standard deviations of pH and aw values and moisture, protein, fat, NaCl, carbohydrate and hydroxyproline contents during curing from day 210 to day 360 are shown in Table 2. The pH remained constant (p > 0.05) and aw and moisture content decreased (p < 0.05) throughout processing. Furthermore, no dier-

Total viable count 4.05 0.06c 3.82 0.12b 3.64 0.10a

Micrococcaceae 3.72 0.09c 3.40 0.01b 3.15 0.13a

Lactic acid bacteria 3.17 0.06c 3.07 0.04b 2.99 0.02a

Enterobacteria ND ND ND

Values in the same column with dierent letters are signicantly different (p < 0.05). ND: not detected.

Table 2 Mean and standard deviation of pH and aw values, moisture (%) and protein, fat, NaCl, carbohydrate and hydroxyproline contents (% DM) of dry-cured cecina at three dierent processing times (210, 270 and 360 days) Days 210 270 360 pH 5.95 0.11 5.86 0.12a 5.84 0.03a
a

aw 0.930 0.001c 0.898 0.003b 0.874 0.001a

Moisture 60.14 0.20c 51.30 0.10b 50.11 0.68a

Fat 15.23 0.71a 16.03 0.20a 16.17 0.72a

Protein 68.98 1.07a 66.12 3.17a 69.05 0.64a

NaCl 14.35 0.04a 14.60 0.03a 15.01 0.71a

Carbohydrate 0.60 0.18a 0.70 0.25a 0.47 0.10a

Hydroxyproline 0.25 0.004a 0.24 0.003a 0.24 0.030a

a,b,c Values in the same column with dierent letters are signicantly dierent (p < 0.05). DM: Dry matter.

374

C. Molinero et al. / Meat Science 80 (2008) 370379

ences (p > 0.05) were found in fat, protein, NaCl, carbohydrate and hydroxyproline contents between batches. The nal results (360 days) for pH and aw obtained were similar to those found by Rubio et al. (2007) in Cecina de Leon that had been cured for 11 months (pH = 5.85 and aw = 0.878). 3.3. Proteolysis and lipolysis-related parameters Table 3 shows the proteolysis and lipolysis-related values of cecina from day 210 to day 360 of processing, during which time AN content increased (p < 0.05) while PN content decreased (p < 0.05); moreover, a negative correlation was established between AN and PN (r = 0.77, p < 0.05). The NPN and AN values found were higher than those obtained by Garca, Dez, and Zumalacarregui (1997) in the same meat product. This might be explained by the shorter processing time (153 days as opposed to 360 days in our study). However, the lipid fractions changed (p < 0.05) during the process and the amount of NL and PL decreased (p < 0.05) while FFA content increased (p < 0.05) from day 210 to day 360. Thus, lipolytic changes aected NL and PL fractions leading to an accumulation of FFA. In fact, negative correlations between NL with FFA (r = 0.78, p < 0.05) and PL with FFA (r = 0.97, p < 0.05) and a positive correlation between NL and PL (r = 0.69, p < 0.05) were found. No results were found in the literature on lipid fractions in cecina, although similar results were reported for French ham at 179 and 273 days by Buscailhon, Gandermer, and Monin (1994), and in Spanish ham cured for 15 months by Motilva, Toldra, Nieto, and Flores (1993). These results suggest that both proteolysis and lipolysis continued after day 210 up to day 360 and could therefore play an important role in the sensorial and textural changes of cecina, as reported by Buscailhon et al. (1994) in ham. Also, a similar tendency for both proteolysis and lipolysis was noted by Cilla et al. (2005) in ham stored for up to 26 months. 3.4. SDSPAGE electrophoresis Fig. 1 shows the electrophoregram obtained by SDS PAGE for the myobrillar proteins from cecina after 210,

270 and 360 days of curing. Protein and peptide molecular weights (MW) were identied according to MW standards. Table 4 shows the relative quantity of the most relevant proteins throughout processing, during which time these proteins underwent various modications. Quite dierent behaviour was observed for the two major myobrillar proteins throughout cecina processing. Myosin heavy chain (MHC), which appeared at 200 kDa, clearly showed a progressive decrease by as much as 56% from day 210 to day 360. However, the relative concentration of actin (45 kDa) decreased by 12%. Research on the eects of storage on meat products (Bechtel & Parrish, 1983) has shown that myosin is more sensitive to processing than actin (45 kDa). Other high MW bands, such as the 171 and 102 kDa ones, also decreased. At the same time as these modications took place, some peptides in the 159

210 days

270 days

360 days

kDa
171 159 102 95 76 66 58

kDa
200 116 97

66

45 44 39 38 32 28 25 23

45

31

21

Fig. 1. SDSPAGE myobrillar protein electrophoregram for dry-cured cecina processed for 7 months (lanes 1 and 2), 9 months (lanes 3 and 4) and 12 months (lanes 5 and 6) and molecular weight standards (lane 7).

Table 3 Proteolysis-related parameters: non-protein nitrogen (NPN), aminoacids (AN) and peptides (PN) and lipolysis-related parameters: neutral lipids (NL), free fatty acids (FFA) and phospholipids (PL) in dry-cured cecina at three dierent processing times (210, 270 and 360 days) Days Parameters related to proteolysisA NPN 210 270 360
a,b,c A B

Parameters related to lipolysisB PN

a

AN
a

NL
c

FFA
b

PL
a

1377.54 4.13 1462.28 18.87b 1479.75 28.57b

523.63 7.02 534.18 2.10a 653.33 29.30b

344.65 9.24 289.24 8.45b 261.64 1.77a

78.93 0.66 79.29 0.07b 77.73 0.16a

12.60 0.30 13.10 0.10b 16.06 0.04c

8.46 0.37c 7.61 0.02b 6.21 0.11a

Values in the same column with dierent letters are signicantly dierent (p < 0.05). Expressed as mg nitrogen/100 g dry matter. Expressed as % of fatty acids of each fraction respect to the total of fatty acid content.

C. Molinero et al. / Meat Science 80 (2008) 370379 Table 4 SDSPAGE of the relevant myobrillar protein bands of dry-cured cecina at three dierent processing times (210, 270, 360 days) MWa (kDa) Days 210 200 171 159 116 102 97 95 76 66 58 45 44 39 38 3230 28 25 23
a

375

The proteolytic pattern agreed with the results for NPN and AN described above (Table 3), both of which showed that the most intense proteolysis took place during the nal period of processing. 3.5. Instrumental measurement of colour Table 5 shows colour parameters at day 210, day 270 and day 360 of processing. Lightness (L*) values decreased (p < 0.05) from day 270 to day 360 of processing due, in all probability to the decrease in water content. Estevez, Morcuende, and Cava (2003) and Sanabria, Martn Alvarez, and Carrascosa (2004) reported that moisture loss raised pigment concentrations such as myoglobin and caused a reduction in this parameter. L* values also fall in meat products as salt concentration increases, as reported by Fernandez-Lopez, Sayas-Barbera, Perez-Alva (2003). The L* had a positive correz, and Aranda-Catala relation with moisture (r = 0.48, p < 0.05). The parameter a* (redness) increased (p < 0.05) from 210 days to 360 days. Fernandez-Lopez et al. (2003) and Perez-Alvarez et al. (1997) found that an increase in a* values could be caused by salt in dry-cured products. They oered two possible explanations for the eect of salt on the a*: (1) extraction by salt of sarcoplasmic proteins (including myoglobin) towards the meat surface, thereby increasing the presence of compounds that contribute to a red colour on the surface; (2) an increase in water retention capacity caused by the addition of salt could reduce the water content at the meat surface and thus increase concentrations of myoglobin. On the other hand, no signicant (p > 0.05) variation in the mean values of b* (yellowness) were found throughout the process. It may be that the low percentage of fat in cecina means that there were little or no modications due to oxidation that aected b*. 3.6. Instrumental measurement of texture With respect to the instrumental parameters, hardness, chewiness and springiness (Table 5) all increased (p < 0.05) whereas cohesiveness decreased (p < 0.05) between day 210 and day 360 of processing. Changes in hardness and chewiness during curing have been attributed to both the state of the proteins and the water content (Monin et al., 1997). In the nal stage, moisture loss from 60.14% to 50.11% could be enough to cause an increase in

270 11.67 0.63 9.68 1.80 23.25 0.76 0.34 0.06 2.29 0.26 0.72 0.15 4.19 0.72 2.28 0.38 3.86 0.12 1.51 0.15 27.40 0.28 4.13 0.10 2.81 0.13 3.87 0.11 1.50 0.21 1.70 0.14 0.78 0.20 0.23 0.03

360 7.27 0.87 7.99 0.19 30.36 4.38 0.35 0.02 1.83 0.63 0.23 0.12 4.86 0.77 1.79 0.24 4.19 0.13 1.47 0.43 25.94 1.45 3.55 0.16 2.60 0.31 1.97 0.43 0.99 0.01 0.60 0.34 0.14 0.04

16.19 0.69 11.55 1.28 20.81 2.25 2.28 0.42 0.52 0.10 1.37 0.18 1.62 0.18 1.81 0.07 1.46 0.11 29.59 4.58 4.11 0.27 3.15 0.15 3.08 0.11 1.20 0.14 1.00 0.28 0.54 0.26 0.59 0.01

MW: molecular weight.

66 kDa range increased, that might be related to the proteolysis of myosin and other high MW proteins. Several researchers have found similar changes to those described in myobrillar proteins during meat product processing. Thorainsdottir, Arasonm, Geirsdotir, Bogason, and Kristbergsson (2002) reported increases in the intensity of two protein bands (147 kDa and 86 kDa), coupled with lower MHC band intensities. They hypothesised that these protein bands were part of the myosin molecule and came from denaturation of the protein. Larrea, Hernando, Quiles, Lluch, and Perez-Munuera (2006) observed that in dry-cured ham the band at 97 kDa (phosporilase B) disappeared, probably due to a specic sensitivity to salt. Gar ca et al. (1997) studied the changes in proteins during the ripening of Spanish dried beef cecina and also observed changes in the myobrillar proteins: disappearance of MHC and troponin C from the smoking stage and appearance or increases during ripening of three components with MWs of 75, 70 and 65 kDa. Several proteins such as b-tropomyosin, the three subunits of troponin and the myosin light chains are located below 45 kDa. All showed a decrease in intensity throughout processing, mainly during the last period of curing from day 270 to day 360 (Table 4).

Table 5 Results of instrumental measurement of colour and texture in dry-cured cecina (mean standard deviation) at three dierent processing times (210, 270 and 360 days) Days Instrumental measurement of colour L* 210 270 360
a,b,c

Instrumental measurement of texture b* Hardness (g)

a

a*
b

Chewiness (g)
a

Springiness
a

Cohesiveness 0.49 0.01b 0.43 0.05a 0.42 0.01a

26.44 1.43 27.14 1.29b 20.61 0.80a

6.70 1.20 14.93 1.22b 12.43 2.16b

3.98 1.60 4.08 1.41a 6.32 1.43a

1964.16 361.10 2699.05 95.99b 3074.35 464.34c

516.64 155.99 709.27 135.91b 892.66 144.22c

0.52 0.06 0.63 0.11ab 0.68 0.05b

Values in the same column with dierent letters are signicantly dierent (p < 0.05).

376

C. Molinero et al. / Meat Science 80 (2008) 370379 2.83 0.31a 3.34 0.47ab 3.55 0.07b 2.91 0.31a 3.34 0.13b 3.33 0.23b 210 270 360
a,b,c

Flavour parameters

Table 6 shows the sensorial parameters at day 210, day 270 and day 360 of processing. With respect to the visual parameters, changes in marbling, yellowness and intermuscular fat were not detected (p > 0.05); nevertheless, colour homogeneity increased between day 210 and day 360 and colour intensity increased from day 210 to day 270 and then remained unchanged up until day 360 of processing. These results agreed with instrumental measurement of colour, and a positive correlation was found between a* and colour intensity (r = 0.59, p < 0.05). Besides, a signicant negative correlation was established between moisture content and colour homogeneity (r = 0.88, p < 0.05) and between moisture content and colour intensity (r = 0.89, p < 0.05). Similar behaviour in colour parameters were noted by Cilla et al. (2005) in ham throughout extended ripening over 1226 months. Odour intensity increased (p < 0.05) from day 210 to day 270 and then remained unchanged until day 360 of processing. Flavour intensity and aftertaste increased (p < 0.05) from day 210 to day 360 of processing. These results could be related to the changes in proteolysis and lipolysis between day 210 and day 360 of processing. In fact, avour intensity correlated positively with NPN (r = 0.67, p < 0.05), with AN (r = 0.88, p < 0.05) and with FFA (r = 0.84, p < 0.05). Also, odour intensity was positively related to NPN (r = 0.63, p < 0.05). These results are in line with those reported by Toldra (2006) who noted that when the ripening/drying time was lengthy, a relatively higher level of enzyme activity took place. As a consequence, higher free amino acid and FFA amounts were generated, which contributed directly to taste, and indirectly to aroma compounds. Sensorial hardness was in line with the results obtained from instrumental measurement of texture (r = 0.60, p < 0.05). These parameters signicantly increased (p < 0.05) from day 210 to day 360 of processing, however, no changes (p > 0.05) in chewiness were detected, which was not the case for the instrumental measurements. Juiciness decreased (p < 0.05) and pastiness increased from day 210 to day 360 of processing. The decrease in juiciness could be due to water loss throughout processing. Moisture content had a positive correlation with juiciness (r = 0.74, p < 0.05) and a negative one with pastiness (r = 0.72, p < 0.05). Some authors (Careri et al., 1993; Guerrero, Gou, & Arnau, 1999) have

Pastiness Juiciness Table 6 Sensorial parameters (mean standard deviation) of dry-cured cecina at three dierent processing times (210, 270 and 360 days) Texture parameters Chewiness Odour intensity Hardness Odour

3.7. Sensory evaluation

1.75 0.35a 2.46 0.10b 3.72 0.21c Values in the same column with dierent letters are signicantly dierent (p < 0.05). 2.25 0.20a 3.01 0.14b 3.85 0.14c 2.42 0.43a 3.72 0.29b 3.66 0.23b 2.33 0.23a 2.28 0.21a 2.30 0.44a 1.58 0.31a 1.41 0.43a 1.56 0.09a 2.83 0.31a 2.44 0.34a 2.22 0.63a

Visual parameters

Days

Colour homogeneity

Colour intensity

Marbling

Yellowness

Intermuscular fat

2.37 0.58a 2.54 0.44a 2.74 0.40a

3.44 0.04c 3.04 0.03b 2.44 0.02a

2.33 0.26a 2.52 0.32a 3.05 0.31b

2.88 0.36a 3.09 0.13a 3.72 0.21b

Flavour intensity

hardness. In fact, a negative correlation was found between moisture and hardness (r = 0.62, p < 0.05). Besides, it was observed that the moisture content had a negative correlation with chewiness (r = 0.72, p < 0.05) and springiness (r = 0.68, p < 0.05) and a positive correlation with cohesiveness (r = 0.94, p < 0.05). A similar tendency in these texture parameters was found by Buscailhon et al. (1994) in French cured ham, when comparing hams processed for 179 and 273 days.

Aftertaste

C. Molinero et al. / Meat Science 80 (2008) 370379

377

related the degree of proteolysis to pastiness in ham. Their ndings that hams with a higher percentage of NPN had higher scores for pastiness agrees with our research, which established a positive correlation between NPN and pastiness (r = 0.73, p < 0.05). With respect to the results of the triangle tests (Table 7), consumers distinguished (p < 0.01) between cecina processed for 210, 270 and 360 days; nevertheless, it must be emphasized that consumer evaluations of preferences showed signicant dierences (p < 0.05) for products cured for short lengths of time. In fact, the least appreciated ceci-

na (27%) was that processed for 7 months and no signicant dierences were noted for cecina after 9 and after 12 months of processing (p > 0.05). According to consumers, this preference was due to the higher colour and odour intensity. These results agreed with those obtained by the trained panel (Table 6) and instrumental colour measurements (Table 5). 3.8. Principal components analysis Physicochemical, microbiological and sensorial parameters were subjected to principal component analysis (PCA) (Fig. 2). The plot of the two principal components explained 70.65% of the total variance. Fig. 2 shows the loading plot of the dierent variables (A) and the score plot of the samples (B). The rst component (PC1) explained 61.15% of the variance. Microbial counts, moisture and aw, PN and phospholipids had high positive loadings on component 1, whereas NaCl, NPN, AN, FFA and most sensorial textural parameters had negative loadings on component 1. The second component (PC2) accounted for 9.50% of total variance. Total

Table 7 Discriminant analysis between dry-cured cecina processed for 210, 270, and 360 days Compared samples 210 and 270 days 210 and 360 days 270 and 360 days Number of correct judgements 65 66 76 Signicance level 0.01 0.01 0.01

Number of correct judgements in triangle tests (n = 100).

0.5 0.4 0.3 0.2

PC2 9.50 %

A
Yellowness Intermuscular fat Flavour intensity Total viable count AN Sensorial hardness Moisture Cohesiveness Chewiness NaCl FFA Springiness Sensorial chewiness PN PL aw Colour homogeneity After taste Micrococcaceae Pastiness Hardness Lactic acid bacteria Juiciness NPN pH Marbling Olour intensity NL Colour intensity b * a* L*

0.1 0 -0.1 -0.2 -0.3 -0.4 -0.5 -0.5

-0.4

-0.3

-0.2

-0.1

0.1

0.2

0.3

0.4

0.5

PC1 61.15 %

8 6 4
PC2 9.50 %

2 0 -2 -4 -6 -8 -8 -6 -4 -2 0
PC1 61.15 %

Fig. 2. Loading plot (A) and score plot (B) after principal component analysis of physicochemical and sensory characteristics of dry-cured cecina processed for 210 days (d), 270 days (j) and 360 days (N).

378

C. Molinero et al. / Meat Science 80 (2008) 370379 Bligh, E. G., & Dyer, W. J. (1959). A rapid method for total lipid extraction and purication. Canadian Journal of Biochemistry and Physiology, 37, 911917. BOCyL (1994). Order of 17th January 1994, by which the Regulation of Specic Denomination Cecina de Leon and its Regulatory Board. Ocial Bulletin of Castile and Leon, (21/1/1994), pp. 444450. Bonnet, M., & Kopp, J. (1984). Dosage du collagene dans les tissus conjonctifs, la viande et les produits carnes. Cahiers Techniques du INRA, 5, 1930. Breene, W. M. (1975). Application of texture prole analysis to instrumental food texture evaluation. Journal of Texture Studies, 6, 5382. Buscailhon, S., Gandermer, G., & Monin, G. (1994). Time related changes in intramuscular lipids of French dry cured ham. Meat Science, 37, 245255. Careri, M., Mangia, A., Barbieri, G., Bolzoni, L., Virgili, R., & Parolari, G. (1993). Sensory property relationships to chemical data of Italiantype dry-cured ham. Journal of Food Science, 58, 968972. CIE (1976). International Commission on Illumination, Colorimetry: ocial recommendations of the International Commission on Illumination. Publication CIE No. 15 (E-1.3.1) Paris, France: Bureau Central CIE. Cilla, I., Martnez, L., Beltran, J. A., & Roncales, P. (2005). Factors aecting acceptability of dry cured ham throughout extended maturation under bodega conditions. Meat Science, 69, 789795. Cordoba, J. J., Antequera Rojas, T., Garca Gonzalez, C., Ventanas Barroso, J., Lopez-Bote, C., & Asensio, M. A. (1994). Evolution of free amino acids and amines during ripening of Iberian cured ham. Journal of Agricultural and Food Chemistry, 42, 22962301. De Ketelaere, A., Demeyer, D., Vandekerckhove, P., & Vervaeke, I. (1974). Stoichiometry of carbohydrate fermentation during dry sausage ripening. Journal of Food Science, 39, 297300. Estevez, M., Morcuende, D., & Cava, R. (2003). Oxidative and colour changes in meat from three lines of free-range reared Iberian pigs slaughtered at 90 kg live weight and from industrial pigs during refrigerated storage. Meat Science, 65, 11391146. Fernandez-Lopez, J., Sayas-Barbera, E., Perez-Alvarez, J. A., & Aranda Catala, V. (2003). Eect of sodium chloride, sodium tripolyphosphate and pH on color properties of pork meat. Industrial Applications: Colour Research and Application, 29, 6774. Garca, I., Dez, V., & Zumalacarregui, J. M. (1997). Changes in nitrogen fractions and free amino acids during ripening of Spanish dried beef: Cecina. Journal of Muscle Foods, 9, 257266. Garca-Regueiro, J. A., Gilbert, J., & Daz, I. (1994). Determination of neutral lipids from subcutaneous fat of cured ham by capillary gas chromatography and liquid chromatography. Journal of Chromatography, 667, 225233. Guerrero, L., Gou, P., & Arnau, J. (1999). The inuence of meat pH on mechanical and sensory textural properties of dry cured ham. Meat Science, 52, 267273. ISO (International Organization for Standardization) (1973). Determination of total fat content, ISO 1443:1973 standard. In International standards meat and meat Product-Geneva. Switzerland: International Organization for Standardization. ISO (International Organization for Standardization) (1978). Determination of nitrogen content, ISO 937:1978 standard. In International standards meat and meat Product-Geneva. Switzerland: International Organization for Standardization. ISO (International Organization for Standardization) (1996). Determination of chloride content Part 1: Vollhard method, ISO 18411:1996 standard. In International standards meat and meat ProductGeneva. Switzerland: International Organization for Standardization. ISO (International Organization for Standardization) (1997). Determination of moisture content, ISO 1442:1997 standard. In International ` standards meat and meat Product-Geneva. Switzerland: International Organization for Standardization. Laemmli, U. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680685.

viable count, AN and some of the sensorial parameters such as yellowness, intermuscular fat, and avour intensity presented positive loadings on this component. The highest negative coecients on component 2 turned out to be NL, instrumental parameters of colour (L*, a*and b*), sensorial colour and odour intensity. According to PCA, the samples were distributed on the two rst PC in three clearly separated groups. PC1 separated the three types of cecina. PC2 also separated cecina processed for 270 days from cecina processed for 360 and for 210 days. Cecina processed for 210 days was grouped together on the right-hand side quadrant of the PC1 plot. The position of cecina processed for 210 days on the PC plot corresponded to high moisture content and aw values, microbial counts, PN, NL and PL, cohesiveness and juiciness. The cecina after 360 days of processing had negative loadings on PC1 and positive ones on PC2 that corresponded to high levels of NaCl, AN, FFA and some sensorial parameters such as avour intensity, aftertaste, colour homogeneity and pastiness. 4. Conclusion The results demonstrated that the properties of cecina underwent various modications when processing was extended up to 360 days. Protein and lipid fractions underwent the main changes, which aected the sensory properties. Curing time improved sensory quality up to day 270; however, longer periods of curing did not increase consumer preferences for the product. Acknowledgments The authors would like to thank the laboratory sta of the Estacion Tecnologica de la Carne (Guijuelo) for their technical assistance and the panellist for their participation in the sensory analysis. The assistance oered by Carlos I. Sanchez PhD in this research is also gratefully acknowledged. Our thanks also goes to the Protected Geographical Indication Cecina de Leon for providing us with the samples. This research was supported by the FEDER project and the Institu to Tecnologico Agrario de Castilla y Leon (ITACYL) (No. PEP-2004.001229). Cristina Molinero gratefully acknowl edges a grant provided by the Junta de Castilla y Leon-Spain. References
Arnau, J., Guerrero, L., & Gou, P. (1997). Eect of temperature during the last month of ageing and of salting time on dry cured ham aged for six months. Journal Science Food and Agriculture, 74, 193198. B.O.E. (1979). Order of 31st July 1979, which establishes the ocial methods of analysis of oils and fats, meat products, cereals and related products, fertilisers, phytosanitary products, dairy products, animal feed, water and grape-related products. Ocial Bulletin of State, pp. 2022120247. Bechtel, P. J., & Parrish, F. C. Jr. (1983). Eects of postmortem storage and temperature on muscle protein degradation: Analysis by SDS gel electrophoresis. Journal of Food Science, 48, 294297.

C. Molinero et al. / Meat Science 80 (2008) 370379 Larrea, V., Hernando, I., Quiles, A., Lluch, M. A., & Perez-Munuera, I. (2006). Changes in proteins during Teruel dry-cured ham processing. Meat Science, 74, 586593. Monin, G., Marinova, P., Talmant, A., Martin, J. F., Cornet, M., Lanore, D., et al. (1997). Chemical and structural changes in dry-cured hams (Bayonne hams) during processing and eects of the dehairing technique. Meat Science, 47, 2947. Moore, S., & Stein, W. H. (1954). A modied ninhydrin reagent for the photometric determination of amino acids and related compounds. Journal Biological Chemistry, 211, 907913. Morrison, W. R., & Smith, L. M. (1964). Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron uoridemethanol. Journal of Lipid Research, 5, 600608. Motilva, M. J., Toldra, F., Nieto, P., & Flores, J. (1993). Muscle lipolysis phenomena in the processing of dry cured ham. Food Chemistry, 48, 121125. Muroya, S., Nakajima, I., Oe, M., & Chikuni, K. (2006). Dierence in postmortem degradation pattern among troponin T isoforms expressed in bovine longissimus, diaphragm, and masseter muscles.. Meat Science, 72, 245251. Parolari, G. (1994). Taste quality of Italian raw ham in a free choice prole study. Food Quality and Preference, 5, 129133. Perez-Alvarez, J. A., Sanchez-Rodrguez, M. E., Fernandez-Lopez, J., Gago-Gago, M. A., Ruiz-Peluo, M. C., Rosmini, M. R., et al. (1997). Chemical and colour characteristics of lomo embuchado during salting seasoning. Journal of Muscle Foods, 8, 395411. Rubio, B., Martnez, B., Gonzalez-Fernandez, C., Garca-Cachan, M. D., Rovira, J., & Jaime, I. (2007). Eect of modied atmosphere packaging on the microbiological and sensory quality on a dry cured beef product: Cecina de Leon. Meat Science, 75, 512522.

379

Sanabria, C., Martn-Alvarez, P. J., & Carrascosa, A. V. (2004). Colour and moisture changes during the manufacture of Iberian dry-cured ham caused by some biotic and abiotic factors. Food Science and Technology International, 10, 269275. Thorainsdottir, K. A., Arasonm, S., Geirsdotir, M., Bogason, S. G., & Kristbergsson, K. (2002). Changes in myobrilar proteins during processing of salted cod (Gadus morhua) determined by electrophoresis and dierential scanning calorimetry. Food Chemistry, 77, 327335. Toldra, F. (2006). The role of muscle enzymes in dry-cured meat products with dierent drying conditions. Trend in Food Science Technology, 17, 164168. UNE. (1992). Metodologa prueba triangular. Norma espanola 87006. Asociacion Espanola de Normalizacion y Certicacion (AENOR). n n. UNE. (1995). Ensayo de clasicacio por ordenacio Norma espan 87023. ola n n n Asociacio Espan ola de Normalizacio y Certicacio (AENOR). UNE. (1995). Analisis sensorial. Gua general para la seleccion, entrenamiento y control de evaluadores. Norma espanola 87024-1. UNE. (1996). Analisis sensorial. Gua general para la seleccion, entrenamiento y control de los expertos. Norma espanola 87024-2. Vilar, I., Garca-Fontan, M. C., Prieto, B., Tornadijo, M. E., & Carballo, J. (2000). A survey on the microbiological changes during the manufacture of dry-cured lacon, a Spanish traditional meat product. Journal of Applied Microbiology, 89, 10181026. Virgili, R., Parolari, G., Schivazzappa, C., Soresi Bordini, C., & Borri, M. (1995). Sensory and texture quality of dry-cured ham as aected by endogenous cathepsin B activity and muscle composition. Journal of Food Science, 60, 11831186. Yang, H., Ma, Ch., Qiao, F., Song, Y., & Du, M. (2005). Lipolysis in intramuscular lipids during processing of traditional Xuanwei ham. Meat Science, 71, 670675.